Human rotavirus strain K8 not only possesses a unique VP4 with only 60-70% amino acid sequence homology with other human rotavirus strains but also appears to represent a distinct VP4 serotype in one-way neutralization assays. In previous studies, strain K8 was provisionally classified as VP4 serotype 3, but this remained to be confirmed in reciprocal neutralization tests. To achieve this goal, the cDNA of VP8 (the VP4 serotype-specific subunit) was inserted into the pGEMEX plasmid vector (Promega) and expressed in E. coli (JM109). Neutralization tests with antiserum to the E. coli-expressed K8 VP8 supported the classification of K8 as a distinct serotype. In addition, the cDNAs that represent K8 VP4 or its subunit VP8, or Gottfried VP4 were inserted into a baculovirus expression vector. The cloned gene was expressed at high yield in Spodoptera .frugiperda cells. The guinea pigs immunized with baculovirus expressed VP4 of the K8 strain developed a high level of neutralizing antibodies to the K8 strain. Heterotypic neutralization assays with other human strains are currently underway. Furthermore, the VP8 of human rotavirus strain K8 and the VP4 of porcine rotavirus Gottfried were also expressed in a baculovirus expression vector. Immunization of animals with the expressed proteins is planned.